The invention is generally directed toward signal transduction pathways associated with inflammation, and more particularly toward IL-1/TNF-xcex1-activated kinases (ITAK).
Interleukin-1 (IL-1) and tumor necrosis factor-xcex1 (TNF-xcex1) are two cytokines produced systemically and locally in response to infection, injury or immunological challenge. Based upon studies in which the action of one (or the other) cytokine has been specifically blockaded, or in which purified cytokines have been administered, IL-1 and TNF-xcex1 have been implicated in a number of disease processes. For example, IL-1 has been implicated in inflammatory diseases including rheumatoid arthritis and other degenerative joint diseases, inflammatory bowel disease, type I diabetes, psoriasis, Alzheimer""s disease, and allergy. Overproduction of TNF-xcex1 has likewise been implicated in diseases such as reperfusion injury, rheumatoid arthritis, cardiovascular disease, infectious disease such as HIV infection and HIV-induced neuropathy, allergic/atopic diseases, inflammatory disease/autoimmunity, malignancy, transplant difficulties including organ transplant rejection or graft-versus-host disease, cachexia, and congenital, dermatologic, neurologic, renal, toxicity and metabolic/idiopathic diseases. A particular case where the two cytokines are thought to act synergistically is in the induction of the Systemic Inflammatory Response Syndrome.
Because the consequences of uncontrolled production of IL-1 and TNF-xcex1 can be severe, considerable effort has been expended on therapies that would limit the production or activity of one, or preferably both, of the cytokines. The prevailing therapy has been to administer proteins that bind specifically to the circulating cytokines, thus preventing them from interacting with their cellular receptors. Typically these protein-based therapeutics are antibodies or xe2x80x98solublexe2x80x99 receptors (i.e., recombinant versions of the natural cellular receptors which lack transmembrane and signaling domains). An additional protein-based therapeutic is the IL-1 receptor antagonist protein (IL-1ra), which competes for binding to the same cellular receptors as the agonist forms of IL-1, but does not elicit a cellular signal.
The effectiveness of all three types of protein-based therapy is limited because occupation of even a very small number of IL-1 or TNF-xcex1 receptors by IL-1 or TNF-xcex1 generates a cellular response (and therefore the harmfull effects described above). It is therefore necessary to maintain relatively high levels of anti-cytokine antibody, soluble receptor or antagonist protein in order to drive the equilibrium in favor of complex formation (i.e., to effectively prevent binding of IL-1 or TNF-xcex1 to their respective receptors). Another drawback to such protein-based therapeutics is that each therapeutic is selective for only one of the two cytokines. Thus, large doses of a multitude of therapeutics must be administered to a patient in order to attempt to control IL-1 and TNF-xcex1 production.
Although the biological effects of TNF-xcex1 and IL-1 are quite similar, the structures of the cytokines, and the structure of their receptors, are very different. IL-1 and TNF-xcex1 appear to have overlapping biological activities because the binding of each cytokine to its receptor appears to affect similar post-receptor signal transduction pathways. Many details of these pathways are unclear.
For example, although both cytokines activate the transcription factors NF-xcexaB and AP-1, which leads to the regulated transcription of a wide variety of genes, the particular receptor-proximal effector molecules that regulate this process is unclear. Additionally, both cytokines have been reported to cause the activation of sphingomyelinases and phospholipases that generate, respectively, ceramide and arachidonic acid. Both cytokines also activate members of the mitogen-activated protein kinase (MAPK) family including ERK1, ERK2, and the stress-activated kinases JNK-1 and p38. This family of kinases is activated, to varying extent, by a wide range of hormones, growth factors, heavy metals, protein synthetic inhibitors and ultraviolet light and therefore activation of such kinases cannot be considered unique to the IL-1/TNF-xcex1 signal transduction pathway.
In addition to -items activated by both IL-1 and TNF-xcex1, IL-1 has been reported to specifically activate the IL-1 receptor associated kinase, IRAK, (Cao, Henzel and Gao, Science 271:1128 (1996)). The cytoplasmic domains of TNF receptors have also been reported to interact with other signal transduction molecules such as TRAF1 and TRAF2, FADD, MORT and TRADD. Such TNF-xcex1 receptor-interacting proteins also appear capable of interacting with an extended receptor family, including those that mediate quite distinct cellular responses such as the T- and B-cell activator CD40 and a mediator of apoptosis, fas. (Tewari and Dixit, Curr. Opin. Genet. Dev. 6:39, 1996; Lee et al., J. Exp. Med. 183:669, 1996).
While certain cellular responses may be elicited by IL-1, TNF-xcex1, or other mediators, the only known signaling event that appears to be uniquely induced by IL-1 or TNF-xcex1, but no other defined stimulus, is a protein serine/threonine kinase activity that could be detected in vitro by its ability to phosphorylate xcex2-casein. Guesdon et al., J. Biol. Chem. 268:4236 (1993); Biochem. J. 304:761 (1994). This xcex2-casein kinase activity was induced in fibroblasts and other connective-tissue derived cells by IL-1 and TNF-xcex1 but not by 21 other agents tested. The structure of the xcex2-casein kinase was not elucidated in this report.
However, there has gone unmet a need for substances and/or methods that provide either repression or stimulation of intracellular effects of both IL-1 and TNF-xcex1. There has also gone unmet a need for substances and methods that provide interaction(s) with the post-receptor pathway(s) of IL-1 and TNF-xcex1, as well as substances and methods that provide opportunities to detect agonists and/or antagonists to IL-1 or TNF-xcex1, including single compounds that act as an agonist or antagonist to both IL-1 and TNF-xcex1. The present invention provides these and other related advantages.
The present invention provides nucleic acid and amino acid sequences of protein kinases, preferably human, that interact with at least one post-receptor intracellular pathway of both IL-1 and TNF-xcex1. Such kinases are referred to herein as IL-1/TNF-xcex1-activated kinase (ITAK). Such kinases are induced as enzymatically active kinases capable of phosphorylating specific substrate proteins by treatment of suitable cells with IL-1 or TNF-xcex1. The present invention further provides compositions and methods for the isolation and purification of nucleic acid molecules encoding ITAK. Also disclosed herein are methods for expressing and purifying ITAK, as well as specific assays for the detection of inhibitors or stimulators of ITAK activity, which would have utility as antagonists or agonists of IL-1 and TNF-xcex1.
In addition, the present invention is directed to isolated nucleic acids encoding ITAK and to vectors, including expression vectors, capable of expressing ITAK, preferably from a cDNA encoding ITAK. The present invention includes host cells containing such expression vectors, and processes for producing ITAK by culturing such host cells under conditions conducive to expression of ITAK, and preferably the purification of ITAK, including in industrial quantities. In part due to such purification of ITAK, the invention is also directed to antibodies, preferably monoclonal antibodies, specific for ITAK.
The present invention is also directed to assays utilizing ITAK to screen for potential inhibitors or stimulators of ITAK activity, for example as a means of blocking a signal transduced in response to IL-1 or TNF-xcex1. Further, methods of using ITAK in the design of inhibitors of ITAK activity are also disclosed.
In particular, in one aspect, an isolated nucleic acid molecule encoding an IL-1/TNF-xcex1-activated kinase (ITAK) such as a human ITAK, or variant thereof, is provided. In one embodiment, the isolated nucleic acid molecule comprises the sequence of nucleotides in SEQ ID:NO 1, from nucleotide number 1 to nucleotide number 2940. This isolated nucleic acid molecule encodes a protein having the amino acid sequence of SEQ ID:NO 2. In a related embodiment, nucleic acid molecules encoding ITAK variants are provided, including the Lys81xe2x86x92Ala substituted ITAK variant. Within a related aspect, an isolated ITAK or variant thereof is provided.
Within other related aspects, recombinant vectors, including recombinant expression vectors comprising a promoter operably linked to ITAK-coding sequences are provided. The invention further provides host cells containing any such recombinant vectors.
In still another aspect, the invention provides a nucleic acid probe of at least 15 nucleotides in length which is capable of specifically hybridizing to a nucleic acid sequence encoding an IL-1/TNF-xcex1-activated kinase (ITAK).
Within yet another aspect of the invention, a method of screening for an agent that modulates the kinase activity of an IL-1/TNF-xcex1-activated kinase (ITAK) is provided, comprising: (a) contacting a candidate agent with biologically active ITAK under conditions and for a time sufficient to allow the candidate agent to modulate the kinase activity of the ITAK; and (b) measuring the ability of the candidate agent to modulate the ITAK kinase activity. Within one embodiment, the method further comprises isolating the candidate agent.
Within another aspect of the invention, a method for determining whether a selected agent is an IL-1/TNF-xcex1-activated kinase (ITAK) agonist is provided, comprising: (a) exposing the selected agent to an unstimulated ITAK response pathway under conditions and for a time sufficient to allow a stimulation of the pathway; and (b) detecting stimulation of the response pathway and therefrom determining the presence of an ITAK agonist. Within a related aspect, a method for determining whether a selected agent is an IL-1/TNF-xcex1-activated kinase (ITAK) agonist is provided, comprising: (a) measuring the ITAK kinase activity of an ITAK response pathway; (b) exposing the selected agent to the measured ITAK response pathway; and (c) detecting increased ITAK kinase activity in the response pathway.
Within still another aspect of the invention, a method for determining whether a selected agent is an IL-1/TNF-xcex1-activated kinase (ITAK) antagonist is provided, comprising: (a) exposing the selected agent to an ITAK response pathway in the presence of an ITAK agonist under conditions and for a time sufficient to allow a decrease in stimulation of the pathway; and (b) detecting a decrease in the stimulation of the response pathway relative to the stimulation of the response pathway by the ITAK agonist alone, and therefrom determining the presence of an ITAK antagonist. Utilizing such methods, ITAK agonists and ITAK antagonists are provided.
Within yet another aspect, an ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian xcex2-casein and that can be phosphorylated by isolated ITAK at a rate of at least 40 nmol, preferably at least 80 nmol, even more preferably at least 98 nmol phosphate/mg protein/minute is provided.
Within still other aspects of the invention, a method for detecting IL-1/TNF-xcex1-activated kinase (ITAK) activity is provided, comprising: (a) contacting ITAK with an ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian xcex2-casein in the presence of ATP under conditions and for a time sufficient to allow transfer of a xcex3-phosphate group from an ATP donor to the ITAK phosphorylation substrate peptide acceptor sequence; and (b) measuring the incorporation of phosphate by the ITAK phosphorylation substrate peptide acceptor sequence. Within one embodiment, the ATP is xcex3-(32P)-ATP. In related embodiments of the invention, the ITAK phosphorylation substrate peptide acceptor sequence has the amino acid sequence: Arg-Arg-Arg-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met-His-Gln-Pro-His-Gln. (SEQ ID:NO 3)
Within another aspect of the invention, a method for treating an IL-1- or TNF-xcex1-mediated inflammatory disorder is provided, comprising administering to a patient a therapeutically effective amount of an ITAK antagonist. The invention further provides kits for detecting ITAK in a sample, comprising: an ITAK phosphorylation substrate peptide acceptor sequence that is not mammalian xcex2-casein and that can be phosphorylated by isolated ITAK at a rate of at least 40 nmol, preferably at least 80 nmol, even more preferably at least 98 nmol phosphate/mg protein/minute; and a means for measuring phosphate incorporated by the ITAK phosphorylation substrate peptide acceptor sequence.
The invention further provides methods for identifying gene products that associate with ITAK, comprising: (a) introducing nucleic acid sequences encoding an ITAK polypeptide into a first expression vector such that ITAK sequences are expressed as part of a fusion protein comprising a functionally incomplete first portion of a protein that is essential to the viability of a host cell; (b) introducing nucleic acid sequences encoding a plurality of candidate gene products that associate with ITAK into a second expression vector such that any candidate gene products are expressed as part of a fusion protein comprising a second functionally incomplete portion of the protein that is essential to the viability of the host cell; (c) introducing the first and second expression vectors into a host cell under conditions and for a time sufficient such that host cell survival is dependent upon reconstitution of both the first and second functionally incomplete portions of the protein into a functionally complete protein; and (d) identifying surviving host cells, and therefrom determining the nucleic acid sequences encoding candidate gene products that associate with ITAK in the second expression vector.
In one embodiment of this aspect of the invention, the host cell is a yeast host cell. In another embodiment of this aspect of the invention the yeast is yeast strain Y190. In related embodiments, the protein that is essential to the viability of the host cell is the modular yeast transcription factor GAL4. In another related embodiment the functionally incomplete first portion of a protein that is essential to the viability of the host cell comprises the N-terminal 147 amino acids of the modular yeast transcription factor GAL4, while in another embodiment the functionally incomplete second portion comprises the C-terminal 114 amino acids of the modular yeast transcription factor GAL4. Within yet another embodiment, the functionally incomplete first portion of a protein that is essential to the viability of the host cell comprises the DNA binding domain of a modular transcription factor. In a related embodiment, the functionally incomplete second portion of a protein that is essential to the viability of a unicellular host comprises a transcriptional activation domain of a modular transcription factor.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. Various references are set forth herein that describe certain procedures or compositions (e.g., plasmids, etc.). All references cited herein are incorporated herein by reference in their entirety.